WO2024059258A1 - Nucléosides et phosphoramidites marqués par clic - Google Patents

Nucléosides et phosphoramidites marqués par clic Download PDF

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WO2024059258A1
WO2024059258A1 PCT/US2023/032859 US2023032859W WO2024059258A1 WO 2024059258 A1 WO2024059258 A1 WO 2024059258A1 US 2023032859 W US2023032859 W US 2023032859W WO 2024059258 A1 WO2024059258 A1 WO 2024059258A1
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oligonucleotide
methoxy
nucleotide
hydrogen
cap
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PCT/US2023/032859
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English (en)
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Catherine R. FOWLER
Brian E. REAM
John C. Rohloff
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Somalogic Operating Co., Inc.
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Publication of WO2024059258A1 publication Critical patent/WO2024059258A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • This disclosure relates to the field of click-labeled uridine bases, nucleosides, and phosphoramidites, including improved methods of synthesis, oligonucleotides comprising the click-labeled nucleosides, methods of synthesizing spin-labeled oligonucleotides using click- labeled nucleotides, and spin-labeled oligonucleotides comprising click-labeled nucleosides.
  • NV nitrogen vacancy
  • a compound having the structure , or a salt thereof, is provided. In some embodiments,
  • Xi and X2 are each independently selected from methoxy and hydrogen.
  • X3 is selected from a methoxy, fluoro, hydrogen, and /c/V-butyldimethylsilyloxy.
  • Xi and X2 are both methoxy.
  • X3 is hydrogen.
  • X3 is methoxy.
  • X3 is fluoro.
  • X3 is tert-butyldimethylsilyloxy.
  • a compound provided herein is selected from:
  • a compound having the structure , or a salt thereof is provided.
  • Xi and X2 are each independently selected from methoxy and hydrogen.
  • X3 is selected from a methoxy, fluoro, hydrogen, and tert-butyldimethylsilyloxy.
  • Xi and X2 are methoxy.
  • X3 is hydrogen.
  • X3 is methoxy.
  • X3 is fluoro.
  • X3 is /c/V-butyldimethylsilyloxy.
  • the compound is selected from:
  • a method of producing a compound having the structure: salt thereof is provided.
  • a method of producing a compound having the structure: salt thereof is provided.
  • Xi and X2 are each independently selected from methoxy and hydrogen.
  • X3 is selected from a methoxy, fluoro, hydrogen, and /c/V-butyldimethylsilyloxy.
  • the method comprising reacting the compound or a salt thereof, with 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite and pyridine trifluoroacetic acid in dichloromethane, 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite and diisopropylethylamine in dichloromethane, or similar conditions.
  • the method produces a compound selected from:
  • a method of producing a compound having the structure: , or a salt thereof is provided.
  • Xi and X2 are each independently selected from methoxy and hydrogen.
  • X3 is selected from a methoxy, fluoro, hydrogen, and tert-butyldimethylsilyloxy.
  • the method comprises reacting the compound
  • the method produces a compound selected from:
  • Xi and X2 are each independently selected from methoxy and hydrogen;
  • X3 is selected from a methoxy, fluoro, hydrogen, and /c/V-butyldimethylsilyloxy; and comprising the steps of: a) b) reacting the compound -cyanoethyl N,N,N’,N’- tetraisopropylphosphorodiamidite and pyridine trifluoroacetic acid in dichloromethane, 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite and diisopropylethylamine in dichloromethane, or similar conditions.
  • the method produces a compound selected from:
  • oligonucleotides comprising at least one spin- labeled nucleotide, wherein at least one spin-labeled nucleotide in the oligonucleotide has the structure: wherein W is a functional molecule.
  • X3 is selected from a methoxy, fluoro, hydrogen, and tert-butyldimethylsilyloxy.
  • X4 is selected from OH, -OR’, -SR’, and -Z-P(Z’)(Z”)O-R”, wherein Z, Z’, and Z” are each independently selected from O and S, R’ is H or a cap, and R” is H, a cap, or an adjacent nucleotide.
  • X5 is selected from -O-ss, -OR’, -SR’, and - Z-P(Z’)(Z”)O-R”, wherein ss is a solid support, Z, Z’, and Z” are each independently selected from O and S, R’ is H or a cap, and R” is H, a cap, or an adjacent nucleotide.
  • the solid support is controlled-pore glass (CPG).
  • Z’ is S and Z” is O.
  • Z’ and Z” are O.
  • oligonucleotides comprising at least one spin- labeled nucleotide, wherein at least one spin-labeled nucleotide in the oligonucleotide has the structure: , wherein W is a payload moiety.
  • X3 is selected from a methoxy, fluoro, hydrogen, and tert-butyldimethylsilyloxy.
  • X4 is selected from OH, -OR’, -SR’, and -Z-P(Z’)(Z”)O-R”, wherein Z, Z’, and Z” are each independently selected from O and S, and R is an adjacent nucleotide in the oligonucleotide, R’ is H or a cap, and R” is H, a cap, or an adjacent nucleotide.
  • X5 is selected from -O-ss, -OR’, -SR’, and -Z-P(Z’)(Z”)O-R”, wherein ss is a solid support, Z, Z’, and Z” are each independently selected from O and S, R’ is H or a cap, and R” is H, a cap, or an adjacent nucleotide.
  • the solid support is controlled- pore glass (CPG).
  • Z’ is S and Z” is O.
  • Z’ and Z” are O.
  • a method of producing an oligonucleotide comprising at least one 5-position modified nucleoside comprising synthesizing an oligonucleotide comprising at least one nucleotide having the structure: into a nucleotide sequence on a solid support; and reacting the oligonucleotide with a reagent comprising a payload moiety and an azide moiety.
  • X3 is selected from a methoxy, fluoro, hydrogen, and /c/V-butyldimethylsilyloxy.
  • X4 is selected from OH, -OR’, -SR’, and -Z- P(Z’)(Z”)O-R”, wherein Z, Z’, and Z” are each independently selected from O and S, R’ is H or a cap, and R” is H, a cap, or an adjacent nucleotide.
  • X5 is selected from -O-ss, -OR’, -SR’, and -Z-P(Z’)(Z”)O-R”, wherein ss is a solid support, Z, Z’, and Z” are each independently selected from O and S, R’ is H or a cap, and R” is H, a cap, or an adjacent nucleotide.
  • the solid support is controlled-pore glass (CPG).
  • Z’ is S and Z” is O.
  • Z’ and Z” are O.
  • a method of producing an oligonucleotide comprising at least one 5-position modified nucleoside comprising synthesizing an oligonucleotide comprising at least one nucleotide having the structure: into a nucleotide sequence on a solid support; and reacting the oligonucleotide with a reagent comprising a payload moiety and a tetrazine moiety.
  • X3 is selected from a methoxy, fluoro, hydrogen, and /c/V-butyldimethylsilyloxy.
  • X4 is selected from OH, -OR’, -SR’, and -Z-P(Z’)(Z”)O-R”, wherein Z, Z’, and Z” are each independently selected from O and S, R’ is H or a cap, and R” is H, a cap, or an adjacent nucleotide.
  • X5 is selected from -O-ss, -OR’, -SR’, and -Z-P(Z’)(Z”)O-R”, wherein ss is a solid support, Z, Z’, and Z” are each independently selected from O and S, R’ is H or a cap, and R” is H, a cap, or an adjacent nucleotide.
  • the solid support is controlled-pore glass (CPG).
  • Z’ is S and Z” is O.
  • Z’ and Z” are O.
  • Fig. 1 shows a chromatogram of DBCO-modified oligonucleotide overlaid with the same oligonucleotide clicked to TEMPO-azide (two peaks corresponding to two diastereomers).
  • Fig. 2 shows chromatograms of TCO-modified oligonucleotide (bottom panel), cyanine-3 tetrazine solution (middle panel) and the resulting product of the oligonucleotide clicked to the cyanine-3 tetrazine (top panel).
  • the compounds provided herein allow for the use of copper-free click chemistry reactions, which may have advantages over copper-requiring click reactions such as copper-catalyzed alkyne-azide cycloadditions.
  • omitting the copper catalyst may reduce or eliminate cell toxicity. See, e.g., Jewett et al., Chem Soc Rev, 2010, 39(4), 1272-1279.
  • these copper-free reactions may be easier to control and/or optimize because the reaction involves fewer components.
  • purification may be more straightforward and may be carried out, in some embodiments, by desalting or size exclusion methods.
  • the compounds provided herein comprising a cyclooctyne at the five position of a uracil nucleobase allows for simpler, faster, and/or more readily controlled reactions with a tetrazine-payload in the absence of a copper catalyst.
  • ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the context clearly dictates otherwise).
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • “about” or “consisting essentially of’ mean ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated.
  • the terms “include” and “comprise” are open ended and are used synonymously.
  • nucleotide refers to a ribonucleotide or a deoxyribonucleotide, or a modified form thereof, as well as an analog thereof.
  • Nucleotides include species that include purines (e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs) as well as pyrimidines (e.g., cytosine, uracil, thymine, and their derivatives and analogs).
  • modify dU is used to generally refer to uridylyl nucleotides comprising a 5-position modification.
  • Use of the term “mod dU” is not intended to be limiting with regard to the 2’ position of the ribose, and the term should be construed to include, but not be limited to, nucleotides comprising, for example, -H, -OH, -Ome, or -F at the 2’ -position, unless a particular 2’ moiety is indicated.
  • DBCO dU is used to generally refer to uridylyl nucleotides comprising a 5-position dibenzocyclooctyne.
  • Use of the term “DBCO dU” is not intended to be limiting with regard to the 2’ position of the ribose, and the term should be construed to include, but not be limited to, nucleotides comprising, for example, -H, -OH, - Ome, or -F at the 2’ -position, unless a particular 2’ moiety is indicated.
  • TCO dU is used to generally refer to uridylyl nucleotides comprising a 5-position trans-cyclooctene.
  • Use of the term “TCO dU” is not intended to be limiting with regard to the 2’ position of the ribose, and the term should be construed to include, but not be limited to, nucleotides comprising, for example, -H, -OH, - OMe, or -F at the 2’-position, unless a particular 2’ moiety is indicated.
  • nucleic acid As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide” are used interchangeably to refer to a polymer of nucleotides and include DNA, RNA, DNA/RNA hybrids and modifications of these kinds of nucleic acids, oligonucleotides and polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included.
  • polynucleotide oligonucleotide
  • nucleic acid include double- or single-stranded molecules as well as triple-helical molecules.
  • nucleic acid, oligonucleotide, and polynucleotide are broader terms than the term aptamer and, thus, the terms nucleic acid, oligonucleotide, and polynucleotide include polymers of nucleotides that are aptamers, but the terms nucleic acid, oligonucleotide, and polynucleotide are not limited to aptamers.
  • the term “at least one nucleotide” when referring to modifications of a nucleic acid refers to one, several, or all nucleotides in the nucleic acid, indicating that any or all occurrences of any or all of A, C, T, G or U in a nucleic acid may be modified or not.
  • a “phorphoramidite” is a nucleotide comprising a group attached to the 3’ carbon of the ribose, or an equivalent position on another sugar moiety.
  • a phosphoramidite comprises a protecting group on the 5 ’-OH of the ribose, such as a trityl protecting group, for example, a dimethoxytrityl protecting group.
  • solid phase synthesis refers to solid-phase oligonucleotide synthesis using phosphoramidite chemistry, unless specifically indicated otherwise.
  • click chemistry reaction refers to bio- orthogonal reaction that joins two compounds together under mild conditions in a high yield reaction that generates minimal, biocompatible and/or inoffensive byproducts.
  • a click chemistry reaction requires a copper catalyst.
  • a click chemistry reaction is carried out in the absence of a copper catalyst.
  • a click chemistry reaction is a copper-free reaction.
  • a click chemistry reaction is a copper-free reaction and is promoted, for example, by ring strain.
  • the present disclosure provides the compounds shown in Table A, as well as salts thereof, and methods of making and using the compounds.
  • X3 in the structures in Table A may, in some embodiments, be selected from methoxy, fluoro, hydrogen, and /c/V-butyldimethylsilyloxy.
  • compounds 1 to 4 in Table A may be used in solid-phase oligonucleotide synthesis to produce oligonucleotides comprising one or more spin-labeled nucleotides.
  • the 3’ carbon of the ribose is linked to a solid phase through a linker moiety selected from succinate, diglycolate, and alkylamino.
  • a salt may be formed with a suitable cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as Al +3 .
  • Suitable organic cations include, but are not limited to, ammonium ion (i.e., NEE + ) and substituted ammonium ions (e.g., NFER X+ , NH2R X 2 + , NHR X 3 + , NR X 4 + ).
  • Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperizine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
  • An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .
  • a salt may be formed with a suitable anion.
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
  • suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acety oxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric.
  • Examples of suitable organic acids
  • the terms “modify,” “modified,” “modification,” and any variations thereof, when used in reference to an oligonucleotide means that at least one of the four constituent nucleotide bases (i.e., A, G, T/U, and C) of the oligonucleotide is an analog or ester of a naturally occurring nucleotide.
  • the modified nucleotide confers nuclease resistance to the oligonucleotide. Additional modifications can include backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine, and the like.
  • Nonlimiting exemplary caps include 5 ’-trimethoxy stilbene cap, 5’ pyrene cap, 5’ adenylated cap, 5’ guanosine triphosphate cap, 5’ N7-methyl guanosine triphosphate cap, and 3’ Uaq cap.
  • modifications can include substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, and those with modified linkages (e.g., alpha anomeric nucleic acids, etc.).
  • internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages
  • any of the hydroxyl groups ordinarily present on the sugar of a nucleotide may be replaced by a phosphonate group or a phosphate group; protected by standard protecting groups; or activated to prepare additional linkages to additional nucleotides or to a solid support.
  • the 5' and 3' terminal OH groups can be phosphorylated or substituted with amines, organic capping group moieties of from about 1 to about 20 carbon atoms, polyethylene glycol (PEG) polymers in one embodiment ranging from about 10 to about 80 kDa, PEG polymers in another embodiment ranging from about 20 to about 60 kDa, or other hydrophilic or hydrophobic biological or synthetic polymers.
  • Oligonucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including 2'-O-methyl, 2'-O-allyl, 2'-O-ethyl, 2'-O- propyl, 2'-O-CH2CH2OCH3, 2'-fluoro, 2'-NH2 or 2'-azido, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • analogous forms of ribose or deoxyribose sugars that are generally known in the art, including 2'-O-methyl, 2'-O-allyl, 2'-O-ethyl, 2'-O- propyl,
  • one or more phosphodiester linkages may be replaced by alternative linking groups.
  • alternative linking groups include embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR X 2 (“amidate”), P(O) R x , P(O)OR Xl , CO or CH2 (“formacetal”), in which each R x or R Xl are independently H or substituted or unsubstituted alkyl (C1-C20) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalky, cycloalkenyl or araldyl.
  • Oligonucleotides can also contain analogous forms of carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • a modification to the nucleotide structure can be imparted before or after assembly of a polymer.
  • a sequence of nucleotides can be interrupted by non-nucleotide components.
  • An oligonucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the phosphoramidites are useful for incorporation of the modified nucleoside into an oligonucleotide by chemical synthesis
  • the triphosphates are useful for incorporation of the modified nucleoside into an oligonucleotide by enzymatic synthesis.
  • the compounds provided herein, and in particular, compounds of Table A may be used in standard phosphoramidite oligonucleotide synthesis methods, including automated methods using commercially available synthesizers.
  • the click chemistry moiety on the oligonucleotide can be reacted with a payload reagent modified with a complementary click chemistry moiety to yield mod dU.
  • An exemplary click reaction used in the present disclosure is strain-promoted alkyne-azide cycloaddition.
  • Another exemplary click reaction used in the present disclosure is trans-cyclooctene-tetrazine ligation.
  • Various reagents comprising a payload moiety and an azide moiety or a tetrazine moiety for use in click chemistry are commercially available.
  • Triethylamine (1.3 mL, 9.5 mmol, 3 eq) was added to the stirring mixture, which was transferred to a water bath and was heated under an inert atmosphere at 65°C. Reaction progress was monitored by reversed phase HPLC (Waters 2795 HPLC with a 2489 detector and using a Waters Symmetry column, buffer A: lOOmM triethylammonium acetate, buffer B: acetonitrile, gradient: 70% buffer B, isocratic, over 30 minutes). After stirring approximately 5 hours, analysis showed the reaction to be complete. The mixture was stirred at room temperature an additional 16 hours, when stirring was discontinued and solvent was evaporated to recover a yellowish foam.
  • the crude mixture was applied to a silica gel flash column equilibrated with 1% triethylamine/75% ethyl acetate/ 24% hexanes.
  • the product was initially eluted with the same mobile phase, which was modified as the elution progressed to 99% ethyl acetate/ 1% tri ethylamine and finally 2% methanol/ 97% ethyl acetate/ 1% tri ethylamine to complete the elution.
  • Product-containing fractions were concentrated to provide a white to off-white foam (11.58 g, 91% yield).
  • An ABI 3900 automated DNA synthesizer (Applied Biosystems, Foster City, CA) was used with conventional phosphoramidite methods with minor changes to the coupling conditions for modified phosphoramidites.
  • Modified phosphoramidites were used in 0.1 M solutions using acetonitrile with 0-40% dichloromethane and 0-20% sulfolane as the solvent.
  • Solid support was an ABI style fritted column packed with controlled pore glass (CPG, LGC Biosearch Technologies, Petaluma CA) loaded with 3’-DMT-dT succinate with 1000 A pore size. All syntheses were performed at the 50 nmole scale and the 5’ end of each sequence was modified with a hexaethyleneglycol spacer and biotin group for support attachment.
  • DBCO dU variant was done as a single-base replacement at select sites within the DNA strand using phosphoramidites synthesized according to Example 1. Deprotection was accomplished by treating with concentrated ammonium hydroxide at 55°C for 4-6 hours, the product mixtures were filtered and residual solvents removed in a Genevac HT-12 evaporator. Identity and percent full length product were determined using an Agilent 1290 Infinity with an Agilent 6130B single quadrupole mass spectrometry detector using an Acquity C18 column 1.7 pm 2.1x100mm (Waters Corp, Milford, MA).
  • Each oligonucleotide mixture received an aliquot of the azide solution at a 4: 1 ratio of azide to oligonucleotide (based on synthesis scale) and the resulting mixture was mixed at room temperature for 24 to 65 hours, at which time analysis by LC/MS (Agilent 1290 Infinity, configured as above) confirmed that each reaction had reached a quantitative cycloaddition. See Fig. 1.
  • the resulting products had two stereoisomers which can be observed on the resulting chromatogram as dual peaks. See Fig. 1.
  • Each reaction mixture was then applied to a centrifugal filter (Millipore Amicon Ultra- 15 3K), washed three times with 5 mL WFI per wash for removal of small molecule impurities. Product was collected in approximately 500 pL WFI without further purification.
  • An ABI 3900 automated DNA synthesizer (Applied Biosystems, Foster City, CA) was used with conventional phosphoramidite methods with minor changes to the coupling conditions for modified phosphoramidites.
  • Modified phosphoramidites were used in 0.1 M solutions using acetonitrile with 0-40% dichloromethane and 0-20% sulfolane as the solvent.
  • Solid support was an ABI style fritted column packed with controlled pore glass (CPG, LGC Biosearch Technologies, Petaluma CA) loaded with 3’-DMT-dT succinate with 1000 A pore size.
  • Each oligonucleotide mixture received an aliquot of the cyanine 3 tetrazine solution at a 2: 1 ratio of tetrazine to oligonucleotide and the resulting mixture was mixed at room temperature for minimum 5 minutes, at which time analysis by LC/MS (Agilent 1290 Infinity, configured as above) confirmed that each reaction had reached a quantitative cycloaddition. See Fig. 2.
  • the resulting products had four stereoisomers which may be observed on the resulting chromatogram as multiple peaks. See Fig. 2.

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Abstract

L'invention concerne des bases d'uridine marquées par clic, des nucléosides et des phosphoramidites, comprenant des procédés de synthèse améliorés, des oligonucléotides comprenant les nucléosides marqués par clic, des procédés de synthèse d'oligonucléotides marqués par centrifugation à l'aide de nucléotides marqués par clic, et des oligonucléotides marqués par centrifugation comprenant des nucléosides marqués par clic.
PCT/US2023/032859 2022-09-16 2023-09-15 Nucléosides et phosphoramidites marqués par clic WO2024059258A1 (fr)

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